Hepatitis B virus (or HBV) infects humans at a very high rate. It is estimated that 15% of the U.S. population have been infected, and in some African and Asian countries, as much as 20% of the adult population are contagious chronic HBV carriers, with over 50% infected.
HBV infection is transmitted by three general mechanisms: (1) by inoculation with infected blood or body fluids, either in large amounts (as in blood transfusions) or in small amounts (as in an accidental skinprick); (2) by close family or sexual contact; and (3) by infection during pregnancy, where the mother transmits the virus to her child.
Most HBV infections are subclinical, and recovery from both subclinical and clinical infections is usually complete. However, serious long term consequences occur in some cases: (1) about 5% of acute HBV infections result in chronic HBV infection, with the constant potential for infectivity to others and for serious, debilitating liver disease, and (2) it is likely that past infection with HBV may be partly or even wholly responsible for the initiation of fulminant hepatitis, cirrhosis, and primary liver cancer. For example, the normal incidence of primary liver cancer is 1:100,000, but for chronic HBV sufferers the incidence of that cancer is 1:300.
The widespread occurrence of HBV, together with its virulence and its association with chronic or lethal liver disease, constitutes a clinical problem of considerable importance. At constant risk are: (1) blood recipients, patients undergoing hemodialysis or renal dialysis, and the institutionalized; (2) their families and (3) all health professionals (particularly nurses, surgeons and dentists). Hence, it is of paramount importance that carriers of infective HBV be easily and accurately identified and treated.
Identification of carriers of HBV infection has been previously difficult, due both to the nature of the virus and its infective course. Infective carriers often show no symptoms of infection and cannot be identified by routine medical examination. Direct assay for live virus is hampered by the facts that the virus is at best only very poorly propagated in cultured cells and that it is not infectious to small laboratory animals. However, it is known that HBV infection causes development of antibodies to proteins (also called antigens) which are part of the virus. Therefore, assays have been developed to detect the presence of these antigens--hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), hepatitis B virus e antigen (HBeAg)--or their antibodies.
Unfortunately, the presence of antibodies to HBsAg or HBcAg in the blood may indicate only a past HBV infection and does not necessarily indicate a present potential for HBV infectivity. The third HBV antigen, which may actually be a group of several antigens, hepatitis B virus e antigens, or HBeAgs, (Magnius and Espmark, J. Immunol. 109, pp. 1017-1023 (1972)) may be more useful in such assays. Mounting clinical evidence now suggests: (1) that the presence of HBeAgs in the blood is a definitive marker of contagious HBV infection; (2) that the presence of HBeAgs indicates a particular course of potential HBV-associated liver disease, therefore aiding in prognosis and treatment of that disease; and (3) that the presence of antibodies to HBeAgs signals a favorable prognosis for HBV-associated liver disease.
The problem in using HBeAgs in an assay to pinpoint potential HBV infectivity and to predict the course of liver disease is that it has not been previously possible to produce or to purify useful amounts of HBeAgs or their antibodies in a low cost and efficient manner. As noted above, HBV grows at best very poorly in tissue culture and it does not infect small laboratory mammals. Therefore, conventional means for obtaining these viral antigens are not effective in preparing and isolating sufficient quantities of HBeAgs. Moreover, since such preparations are usually contaminated with large amounts HBcAg, the production of antibodies from them results in a mixture of antibodies to HBcAg and HBeAg. Therefore, such antigen and antibody mixtures are not able to distinguish effectively between HBc- and HBe-containing samples in the various assays and accordingly, such assays are not able to detect unambiguously the presence of HBeAgs. The assays are thus ineffective in detecting infectious carriers of HBV.
Recent advances in recombinant DNA technology have allowed the genes for HBsAg and those for HBcAg to be cloned and their protein products synthesized in bacteria (Burrell et al., Nature 279, pp. 43-47 (1979); Pasek et al., Nature 282, pp. 575-579 (1979); Edman et al., Nature 291, pp. 503-506 (1981)). Therefore, it has been suggested that such technology might provide a means to produce purified hepatitis B virus e antigens, i.e., polypeptides displaying the serological variants of HBeAgs. (see e.g. Edman et al., supra).
Unfortunately, the HBV DNA sequences that code for HBeAgs have not been identified. For example, HBeAg has been variously attributed to the DNA polymerase enzyme of HBV (J. L. Melnick et al., "Approaching The Control Of Viral Hepatitis Type B", J. Infectious Diseases, 133, pp. 210-25 (1976)), an idiotype of IgG (A. R. Neurath and N. Strick, "Host Specificity Of A Serum Marker For Hepatitis B: Evidence That Virus e Antigen Has The Properties Of An Immunoglobulin", Proc. Natl. Acad. Sci. USA, 74, pp. 1702-06 (1977)), a dimer of IgG associated with a small peptide (H. A. Fields et al., "Purification And Partial Characterization Of Hepatitis e Antigen", Infection & Immunity, 20, pp. 792-803 (1978)), associated with lactate dehydrogenase isoenzyme no. 5 (G. N. Vyas et al. "Hepatitis B Virus e Antigen: An Apparent Association With Lactate Dehydrogenase Isoenzyme 5", Science, 198, pp. 1068-70 (1977)), or an antigenic marker on the surface of Dane particles and tubular forms (Neurath et al., J. Gen. Virol., 30, pp. 277-85 (1976)). Accordingly, it has not been previously possible to use recombinant DNA techniques to produce HBeAgs.
It also has been reported that HBeAgs are released from HBV core particles purified from serum (Takahashi et al., J. Immunol. 117, pp. 102-105 (1976)) or similar particles purified from liver (Budkowska et al., J. Immunol. 123, pp. 1415-1416 (1979); Yoshizawa et al., J. Gen. Virol. 42, pp. 513-519 (1979)) by treatment with pronase (Budkowska et al.), pronase and 2-mercaptoethanol (Takahashi et al.), or sodium dodecyl sulphate and 2-mercaptoethanol (Budkowska et al.; Takahashi et al.; Yoshizawa et al.) or by disruption by sonication and by treatment with chaotropic agents or centrifugation in CsCl (H. Ohori et al., "Antigenic Conversion From HBcAg To HBeAg by Degradation Of Hepatitis B Core Particles", Intervirology, 13, pp. 74-82 (1980)). However, these methods are not appropriate for the large-scale production of HBeAgs, especially since the resulting products are a mixture of HBc and HBe antigens. Nor do these reports suggest whether HBeAgs are derived from HBcAg or whether HBeAge are different proteins than HBcAg, but which are buried inside the viral particle in such a way that proteolysis of the HBcAg is either necessary for, or an accident to, releasing the HBeAgs. Therefore, these reports do not overcome the difficulty of employing HBeAgs and their antibodies in effective assays or the problems that have prevented the application of recombinant DNA technology to the production of HBeAgs. Moreover, whether HBeAgs are encoded by parts of the HBcAg gene or by entirely separate genes remains unknown. Accordingly, recombinant DNA technology has not been usefully employed to produce HBeAgs.